Alternative pre-mRNA splicing is a major source of proteomic diversity in higher eukaryotes. This process requires that the splicing machinery (spliceosome) select the correct splice sites within thousands of nucleotides of pre-mRNA sequences. However, exactly how the splice sites are recognized is poorly understood. The overall goal of this research proposal is to understand the sequential three-dimensional interactions that guide 3' splice site selection and promote spliceosome assembly. During the critical early stages of 3' splice site recognition, the essential splicing factor U2AF recognizes the poly-pyrimidine tract (Py-tract) pre-mRNA consensus sequence. In turn, the U2AF/Py-tract provides a distinct molecular surface that promotes association of the U2 snRNP, a core component of the active spliceosome. The specific aims of the proposal are: 1. To elucidate the interactions that enable U2AF to recognize a variety of metazoan Py-tract sequences, as opposed to sequence-specific alternative splicing factors. 2. To investigate the sequential interactions with U2AF that recruit the U2 snRNP to the pre-mRNA. 3. To explore the potential protein-interaction targets of other U2AF-homologous motifs (UHM) found in a wide variety of splicing factors. In particular, we focus on the UHM of a medically-relevant target, Tat-SF1, a cellular cofactor for HIV-1 replication. Many serious human diseases are associated with mis-spliced mRNA variants, including CD44 or BRCA1/BRCA2 in cancers, dystrophin in muscular dystrophy, and ATM in ataxia telangiectasia among others. Moreover, disruption of protein-protein interactions mediated by the Tat-SF1 protein presents a potential therapeutic target for treatment of AIDS patients. In the long term, understanding the key interactions that guide normal splice site recognition will provide a basis to target specific molecular strategies, such as peptidomimetics or anti-sense oligonucleotides, against harmful splice variants.